The steroid hormone estrogen plays a critical role in development and maintenance of female reproductive and mammary tissues, but is also involved in maintenance of cardiovascular, skeletal, and neural function, proliferation of breast cancer cells, and concentration of sperm in the male reproductive tract. Moreover, estrogens and selective estrogen receptor modulators (SERMs) are widely used in regulating fertility, alleviating postmenopausal symptoms, and preventing and treating breast cancer. Although it is widely accepted that the estrogen-occupied receptor mediates its effects by interacting with estrogen response elements (EREs) residing in target genes, a number of estrogen-responsive genes contain no identifiable ERE. To understand how estrogen-responsive genes containing AP-1 and Sp1 sites but lacking EREs respond to hormone treatment, we will study the regulation of the endogenous progesterone receptor (PR) gene. The PR plays a crucial role in reproductive and mammary cell development and fertility and has been used clinically as an indicator of estrogen-responsiveness in human breast cancer tumors. Recent studies from our laboratory indicate that the human PR gene, which lacks an ERE, is controlled by multiple cis elements including an AP-1 site and two Sp 1 sites adjacent to an ERE half site. Thus, the PR gene provides an excellent model to define mechanisms by which target genes lacking an ERE can be regulated by hormone. We will monitor the ordered recruitment of transcription factors and coregulatory proteins to these AP-1 and Sp 1 sites in the endogenous PR gene to delineate the temporal sequence of events required for maintaining basal PR expression and initiating and sustaining high levels of transcription using highly sensitive chromatin immunoprecipitation assays. The cis elements and coregutatory proteins involved in mediating the effects of estrogen and the SERMs 4-hydroxytamoxifen and raloxifene will be determined using in vivo footprinting and chromatin immunoprecipitation experiments. Both mammary and uterine cell lines will be examined to define mechanisms by which these clinically important SERMs mediate their tissue-specific effects. To define mechanisms by which estrogen-responsive genes containing AP-1 and Sp 1 sites are regulated, we will isolate and identify novel coregulatory proteins that associate with AP-1 and Sp 1 sites using agarose gel electrophoresis and mass spectrometry analysis and characterize the effects of these proteins on expression of genes containing AP-1 and Sp 1 sites. The insights gained in these combined studies will help to delineate how estrogens and SERMs regulate expression of genes containing AP-1 and Sp1 sites and enhance our understanding of cellular responsiveness of mammary tumor cells as well as normal tissues such as the uterus and breast to these clinically important pharmaceutical agents.